Introduction

The pedunculopontine (PPN) and the laterodorsal (LDT) tegmental nuclei are two complex brainstem structures characteristically containing cholinergic cells (Mesulam et al. 1983), which form an anatomical continuum in the mesopontine tegmentum and are considered as a functional unit working in an integrated fashion (Mena-Segovia 2016). Some of their functions, like locomotion and motor adaptive control, action selection and reward, are related to their reciprocal connections with basal ganglia structures (Mena-Segovia et al. 2004; Roseberry et al. 2016; Takakusaki et al. 2016; Gut and Winn 2016). Specifically, both the PPN and LDT receive abundant projections from the output nuclei of the basal ganglia together with minor projections from the striatum and lateral globus pallidus (Cornwall et al. 1990; Edley and Graybiel 1983; Rye et al. 1987; Shink et al. 1997; Steininger et al. 1992), all of which are GABAergic. However, despite the fact that GABA actions on the PPN have long been known from electrophysiological and pharmacological studies (Ikeda et al. 2004; Nandi et al 2002; Saitoh et al 2003; Torterolo et al 2002), specific studies about the anatomical distribution of GABA receptors (GABARs) in the PPN or LDT have not been carried out so far.

In addition, intermingled with their characteristic cholinergic neurons, PPN and LDT also comprise abundant glutamatergic and GABAergic cells (Wang and Morales 2009; Mena-Segovia et al. 2009; Luquin et al. 2018). Specifically, some of these non-cholinergic cells seem to be the main target of GABAergic projections from the basal ganglia output nuclei (Grofova and Zhou 1998; Mongia et al. 2015; Sherman et al. 2015; Roseberry et al. 2016; Caggiano et al. 2018). However, little is known about the relative distribution of nigral or pallidal terminations on each of the three neurochemical subpopulations of the PPN and LDT, or about the anatomical localization of postsynaptic GABARs potentially mediating the direct inhibitory control from these projections. As a first step to better understand the interactions between the downstream projections of basal ganglia output nuclei and the PPN and LDT at the cellular level and the neural circuitry within these nuclei, we aimed to anatomically confirm the presence of GABA receptors within the PPN and LDT, and to quantitatively determine their relative distribution among the three cell phenotypes in the two nuclei.

Electrophysiological and pharmacological studies have demonstrated the presence of functional GABAA and GABAB receptors in PPN and LDT (Saitoh et al. 2003; Ikeda et al. 2004; Ulloor et al. 2004; Pal and Mallick 2004; Datta 2007; Heinmiller et al. 2009; Ye and Garcia-Rill 2009; Kohlmeier and Kristiensen 2010; Fogel et al. 2010; Kohlmeier et al, 2013). GABAA receptors (GABAARs) are ligand-gated Cl ion channels structurally assembled from five individual protein subunits which include α1-6, β1-3, γ1-3, δ, ε, π, and τ (Olsen and Sieghart 2009). The most common combination of subunits in GABAARs, however, is triplet α1/β2/γ2, which is detected in various cell types in the CNS (Fritschy et al. 1992; McKernan and Whiting 1996). GABAB receptors (GABABRs) are heterodimers formed by the co-assembly of two subunits, GABAB1 and GABAB2, that couple to G-proteins through second messenger pathways and act as modulators of GABA transmission (Bowery et al. 2002). Whole-brain studies on the distribution of either GABAAR or GABABR subunits reported the presence of specific subunits in the rat PPN or LDT in relation to cell bodies, neural processes or neuropil (Fritschy and Möhler 1995; Margeta-Mitrovic et al. 1999; Pirker et al. 2000). However, a specific analysis of GABAAR or GABABR distribution in the PPN and LDT has not been carried out thus far.

Finally, although the PPN and LDT share many common input structures and efferent target areas, both afferent and efferent projections are often topographically organized at the regional level (Dautan et al. 2016a; Hallanger et al. 1987; Oakman et al. 1995; Woolf and Butcher 1986; ** an upper guard zone of 2 µm and a lower one of variable height (2.5 µm on average). Our counting unit was the equator plane of the cell soma, which is the plane of the cell with most sharp borders and it is normally visible in 1–2 microns thickness at most. A cell was counted if the equator was in focus within the height of the disector, which was automatically signaled by the program, and did not touch the forbidden sides (left and bottom) of the disector frame (Luquin et al. 2018).

The sampling fraction was previously determined in a pilot study so as to ensure that a minimum of 100 cells per case from each neuronal phenotype were counted separately in the PPN and LDT, resulting in a coefficient of error (CE) ≤ 0.1 for each of them. The CE was calculated using Eq. 20 from Gundersen et al. (Gundersen et al. 1999). The number of disectors counted ranged between 4 in the smallest areas, and 46 in the largest ones. The volume (V) of PPN and LDT was calculated following the Cavalieri principle (Gundersen and Jensen 1987) using the formula:

$$V\, = \, \, T\, \times \,a\, \times \,\sum P,$$

where T represents the distance between sections (240 µm; a, is the area per point (0.024 mm2), and ΣP is the sum of points counted. Once the volume was calculated, the cell densities (Nv) were finally estimated using the formula:

$$Nv\, = \,N/V.$$

Results

GABAAR α1 and γ2 subunits and GABAB R2 subunit are expressed in both PPN and LDT

Low to moderate numbers of cells expressing α1-α3 and γ2 GABAAR subunits were found in the PPN (Fritschy and Möhler 1995; Rodríguez-Pallares et al. 2001), which are actually some of the most ubiquitous subunits in the brain. Here, we used the α1 and γ2 subunits (GABAAR α1 and GABAAR γ2, respectively) for GABAAR detection. Regarding GABABR, both R1 and R2 subunits are co-expressed throughout the brain and are required for normal receptor function (Benke et al. 1999; Bettler and Tiao 2006; Charles et al. 2001). Initial trials using antibodies against the two subunits revealed a more robust labeling of the R2 subunit, which was, therefore, selected for GABABR detection.

Coronal brainstem sections were first stained for NADPH-d, a histochemical marker of PPN and LDT cholinergic neurons (Vincent et al. 1983) used to identify the cholinergic subpopulation and delineate PPT and LDT territories, using previously described criteria (Luquin et al. 2017; 2018); secondly, they were immunoreacted against GABAAR α1, GABAAR γ2 and GABAB R2. GABAA α1-immunoreactive cell bodies were observed among NADPH-d stained neurons in both PPN and LDT, immersed within a GABAA α1-positive neuropil (Fig. 1a, a’). Similarly, GABAAR γ2-immunoreactivity (GABAAR γ2-ir, Fig. 1b, b’) and GABAB R2-ir (Fig. 1c, c’) were also observed in a large number of cells and in the surrounding neuropil as well. Thus the expression of the three GABA subunits was confirmed, providing anatomical evidence of the presence of GABAA and GABAB receptors in both PPN and LDT.

Fig. 1
figure 1

GABAAR α1-, GABAAR γ2-, and GABAB R2-immunoreactive cells in the PPN and LDT. Coronal sections from the PPN (left) and LDT (right) dually labeled for NADPH-d as a marker of cholinergic neurons (blue–black staining), and GABAR subunits (brown reaction product). a, a’ GABAA α1-ir was observed in cell bodies in the PPN and LDT and also in the neuropil. b, b’, c, c’ Abundant cells also displayed GABAAR γ2-ir and GABAB R2-ir in both PPN and LDT, immersed within an immunoreactive neuropil. Arrowheads point to neurons with lighter NADPH-d reaction product that also contain brown reaction product, suggestive of dually labeled cells. Scale bar: 100 μm

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Virtually all GABAAR α1-, GABAAR γ2- and GABAB R2-immunoreactive cells are neurons

After confirming the expression of the three GABA receptor subunits in the PPN and LDT, we next investigated whether those subunits were localized in neuronal cell bodies. Triple immunolabeling against the neuronal marker NeuN, ChAT, and each GABAR subunit was carried out in separate series from two animals, using the cholinergic neurons for delineation purposes. Confocal images throughout the complete PPN and LDT territories were obtained and all dually labeled cells were counted. The counts revealed that over 90% of GABAAR α1- and GABAB R2-positive cells were NeuN-positive, whereas ~100% of GABAAR γ2-labeled cells co-expressed NeuN-ir in both the PPN and LDT (Fig. 2, Table 3). Given that all three subunits were present in at least 90% of NeuN-positive cells, we have assumed that GABARs are predominantly expressed in neurons and have thus omitted NeuN-ir from subsequent experiments.

Fig. 2
figure 2

Most GABAAR α1-, GABAAR γ2-, and GABAB R2-immunoreactive cells colocalize NeuN-ir. Confocal images of immunofluorescence labeling in the PPN (a–a”, b–b”, c–c’’) show the high degree of colocalization of either GABAAR α1 (a’’), GABAAR γ2 (b’’) or GABAB R2 (c’’) with NeuN, indicating that the three GABA subunits are almost entirely localized in neurons. All images are the resulting projections of two confocal sections only (0.48 µm each). Scale bars in b” and c”: 20 µm. Scale bar in a”, same as in c”

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Table 3 Percentages of co-expression of GABAR subunits with NeuN- and ChAT-ir in PPN and in LDT
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PPN and LDT cholinergic neurons co-express GABAA and GABAB receptors

The observation of sections dually labeled against NADPH-d and the GABAR subunits suggested that some NADPH-d-stained neurons were also DAB-containing (Fig. 1). To unambiguously determine whether the cholinergic cells in the mesopontine tegmentum express any of the three GABAR subunits and to what extent, dual immunofluorescence labeling against ChAT and each of the subunits was carried out, and all single and dually labeled cells quantified. Remarkably, the analysis revealed that ~50% of the cholinergic cells in the PPN expressed GABAAR α1 subunit (Fig. 3a–a’), whereas only 11% of them expressed this subunit in the LDT (Table 3), difference that was statistically significant (Wilcoxon signed-rank test, p = 0.028, n = 6).

Fig. 3
figure 3

Different degrees of co-expression of ChAT-ir (white) and each of the GABAR subunits (green). a–a’’ Only a subset of ChAT-positive neurons (white) co-expressed the GABAA α1-subunit (green), while others were devoid of GABAA α1-ir (asterisk). b–b’’ All ChAT-positive neurons co-expressed GABAA γ2-ir, while abundant non-cholinergic GABAA γ2-ir neurons were also visible in the PPN (a–b’’). c–c’’ A vast majority of ChAT-positive neurons (white) also expressed GABAB R2-ir (green), while non-cholinergic GABAB R2-positive cells were also observed in the LDT. The asterisk marks a ChAT-positive cell lacking GABAB R2-ir. All images are the resulting projections of two confocal sections only (0.48 µm each). Scale bar: 20 µm

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In contrast to the α1 subunit, all cholinergic cells in the PPN and LDT expressed the GABAAR γ2 subunit (n = 5) (Fig. 3; Table 3b–b’). Finally, GABAB R2 subunit was expressed by > 95% of cholinergic cells in the two nuclei (n = 6) (Fig. 3c–c’, Table 3). These results demonstrate that all cholinergic cells in the mesopontine tegmentum express at least one subtype of GABAAR, and virtually all express the GABABR as well.

PPN cholinergic neurons are preferentially located in the caudal portion of the nucleus (Mena-Segovia et al. 2009; Luquin et al. 2018), whereas, in the LDT, these neurons do not show a differential rostrocaudal distribution (Luquin et al. 2018). Given that GABAAR α1 subunit was only present in a subset of the total cholinergic subpopulation in both nuclei, we investigated whether ChAT/GABAAR α1-positive neurons showed a topographical distribution, being differentially present in the rostral versus caudal portions of each nucleus. The comparison between the percentages of dually labeled cells in the rostral versus caudal halves of the two nuclei showed no statistically significant differences either in the PPN (Wilcoxon signed-rank test p = 0.753) or in the LDT (Wilcoxon signed-rank test p = 0.173).

Stereological counts of single GABAAR- and GABABR-expressing cells in the PPN and LDT

Next, we estimated the total number of cells expressing either GABAA or GABAB receptors in PPN and LDT using unbiased stereological methods (Fig. 1). γ2 is the most abundant subunit in the brain and forms part of the majority of GABAAR subtypes (Essrich et al. 1998). GABAAR γ2-ir was also more robust and visually more abundant than GABAAR α1 in our dually labeled sections. In light of this we opted for the γ2 subunit as the representative subunit for the GABAA receptor. The delineation of PPN and LDT territories was carried out as reported previously (Luquin et al. 2018). In brief, we outlined the area contained within the most peripherally located NADPH-positive cells in each nucleus. In the PPN no distinction was made between the pars compacta and the dissipata. Regarding the LDT, both the LDT proper and the ventral LDT or LDTV (Paxinos and Watson 2005) were considered part of a single LDT.

In our dually labeled sections, the brown cells corresponding to single labeled GABAAR γ2- or GABAB R2-positive cells were readily distinguishable from the blue-purple ones stained for NADPH-d (Fig. 1); thus, all single GABAAR γ2- and GABAB R2-positive cells on one hand, and all NADPH-d-stained cells on the other, were counted in the two nuclei (n = 5; Table 4). The mean total number of NADPH-d positive neurons—indicative of cholinergic cells—was 3390 ± 774 and 3121 ± 550, in the PPN and LDT, respectively. These estimations were similar to those obtained in a previous study where the cholinergic neurons were identified using ChAT-ir (Luquin et al. 2018). Regarding the GABARs, the mean total number of non-cholinergic cells expressing GABAAR γ2 (9850 ± 1800) was significantly higher than that of cells expressing GABAB R2 in the PPN (7310 ± 1970; Mann–Whitney U test, p = 0.047 n = 5; Table 4). In contrast, the mean total number of GABAB R2-positive cells in the LDT was slightly higher than that of GABAAR γ2 (9170 ± 1900 and 8280 ± 960, respectively), but not statistically significant (Mann–Whitney U test, p = 0.465; n = 5).

Table 4 Estimated mean number of NADPH-d stained, GABAAR γ2-, and GABAB R2-immunoreactive cells and cell densities in the PPN and LDT
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Co-expression of γ2 and R2 subunits in the GABAergic subpopulation of PPN and LDT

PPN and LDT contain both glutamatergic and GABAergic cells in addition to the cholinergic ones (Wang and Morales 2009; Luquin et al. 2018). Once we had estimated the mean total number of γ2 and R2-expressing non-cholinergic cells, we investigated the neurochemical phenotype of GABAAR- and GABABR-expressing neurons in both nuclei. To determine the number of GABAergic cells expressing each of the two subunits, ISH against the GABA synthetic enzyme GAD67 was carried out in combination with dual immunofluorescence against either GABAAR γ2 (n = 7) or GABAB R2 (n = 5) and ChAT. The semi-quantitative analysis of all dually labeled cells showed a higher percentage of GABAergic cells expressing GABAAR γ2 in the PPN than in LDT (77% ± 8.9 and 49% ± 9.8, respectively; Fig. 4, Table 5), difference that was statistically significant (Wilcoxon signed-rank test, p = 0.018; Fig. 4e). In contrast, the percentage of GAD67-positive cells expressing the GABAB R2 subunit was similar in the two nuclei, representing approximately two-thirds of the total GABAergic subpopulation of PPN and LDT (64% ± 8.7 and 62% ± 16.8, respectively; Fig. 4e, Table 5). Together, these results reveal that there are at least two different subpopulations of GABAergic neurons with respect to their GABAAR and GABABR profiles; in addition, they also revealed that the proportion of GABAergic cells containing the γ2 subunit is different in the PPN and LDT.

Fig. 4
figure 4

Colocalization of GABAA γ2- and GABAB R2-ir with GAD67 mRNA expression in the PPN and LDT. a–a” The majority of GAD67-positive neurons (red) in the PPN co-expressed the γ2 subunit (white). An asterisk marks a single GAD67-positive and γ2-negative neuron in the field (a–a”). b–b” In the LDT, however, only approximately half of the GAD67-positive neurons (red) co-expressed the γ2 subunit (white). Asterisks mark GAD67-positive/γ2-negative cells. c–c”, d–d” almost two-thirds of GABAergic cells co-expressed the R2 subunit in both the PPN and LDT. Asterisks in c–c” and d–d” mark GAD67-positive/R2-negative neurons in the confocal images. All images are the resulting projections of two confocal sections only (0.48 mm each). Scale bars: 20 µm. e Graph showing the percentage of colocalization of GAD67 and the two GABA R subunits in the PPN and LDT. The asterisk indicates a statistically significant difference between γ2-expressing GABAergic cells in the PPN and LDT

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Table 5 Percentages of co-expression of GABAR subunits in the GABAergic and glutamatergic subpopulations of PPN and LDT
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Lastly, we investigated whether regional differences were present in the distribution of GAD67-positive cells expressing either GABAARs or GABABRs. To do so, the percentage of dually labeled cells among GAD67-positive cells in the anterior portion of PPN and LDT was compared to that in the posterior portion. Regarding GAD67/γ2-positive cells, no statistically significant differences were found between the rostral and caudal portions of either PPN (Wilcoxon signed-rank test p = 0.735) or LDT (Wilcoxon signed-rank test p = 0.398). Similarly, no statistically significant differences were found in the rostrocaudal distribution of GAD67/GABAB R2-positive cells either in the PPN (Wilcoxon signed-rank test p = 0.225) or LDT (Wilcoxon signed-rank test p = 0.686). These results indicate that, although GABAergic cells are preferentially located in the rostral portion of both PPN and LDT (Mena-Segovia et al. 2009; Luquin et al. 2018), there are no regional rostrocaudal differences in the relative distribution of GABAergic cells co-expressing either GABAAR γ2 or GABAB R2.

Co-expression of γ2 and R2 subunits in the glutamatergic subpopulation of PPN and LDT

Next we analyzed the glutamatergic subpopulation; in this case, ISH against the VGlut2 was carried out in combination with dual immunofluorescence against either GABAAR γ2 or GABAB R2 and ChAT (n = 5) to determine the number of glutamatergic cells expressing each GABAR subunit. The semi-quantitative analysis of all dually labeled cells revealed that 90% ± 3.7 of glutamatergic cells in the PPN expressed GABAAR γ2 whereas only 65% ± 10.1 of those in the LDT did (Table 5). The statistical comparison between the two showed a significantly higher proportion of co-expression in the PPN (Wilcoxon signed-rank test, p = 0.043; Fig. 5e, Table 5). Regarding the GABAB R2 subunit, the same proportion of glutamatergic cells expressed this subunit in the PPN and LDT, which represented approximately two-thirds of the total (61% ± 12.5 and 61% ± 6.4, respectively; Fig. 5e, Table 5). Thus, two subpopulations of glutamatergic neurons can be distinguished with regard to their GABAAR γ2 and GABAB R2 contents; also, in line with the observations on the GABAergic cells, the proportion of GABAAR γ2-expressing glutamatergic cells was different in the PPN and LDT. Finally, comparisons were also carried out between the respective percentages of glutamatergic cells expressing GABAARs and GABABRs in the anterior versus posterior portions of the PPN and LDT. Regarding VGlut2/γ2-positive cells, no statistically significant differences were found between the rostral and caudal portions of either the PPN (Wilcoxon signed-rank test p = 0.080) or LDT (Wilcoxon signed-rank test p = 0.138). Similarly, no statistically significant differences were found in the rostrocaudal distribution of VGlut2/GABAB R2-positive cells either in the PPN (Wilcoxon signed-rank test p = 0.345) or LDT (Wilcoxon signed-rank test p = 0.500).

Fig. 5
figure 5

Colocalization of GABA subunit γ2 and GABAB R2 with Vglut2 mRNA expression in the PPN and LDT. a–a” The vast majority of VGlut2-positive neurons (red) co-expressed the γ2 subunit (white) in the PPN. The asterisk marks a single VGlut2-positive/γ2-negative neuron (a–a”). b–b” About two-thirds of VGlut2-positive neurons (red) also co-expressed the γ2 subunit (white) in the LDT. An asterisk marks a Vlgut2-positive neuron clearly lacking the R” subunit. c–c”, d–d” about two-thirds of Vglut2-positive neurons co-expressed the R2 subunit both in the PPN and LDT. Asterisks mark Vglut2-positive neurons lacking the GABA receptor subunit. All images are the resulting projections of two confocal sections only (0.48 mm each). Scale bar: 20 µm. e Graph showing the percentage of colocalization of VGlut2 and the two GABA R subunits in the PPN and LDT. The asterisk indicates a statistically significant difference between γ2-expressing glutamatergic cells in the PPN and LDT

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These results indicate that, although glutamatergic cells are preferentially located in the caudal portion of the PPN (Luquin et al. 2018), there are no regional rostrocaudal differences in the relative distribution of glutamatergic cells co-expressing either GABAAR γ2 or GABAB R2 in this nucleus, and the same stands for the LDT.

Relative expression of γ2- and R2 subunits in PPN and LDT

Previous studies from our laboratory (Luquin et al. 2018) estimated the relative composition of PPN and LDT with respect to their three different neurochemical cell subpopulations (Fig. 6, outermost circular crown). This result, together with the present data regarding the extent of co-expression of GABAAR γ2 subunit and GABAB R2 subunit in each of the three cell phenotypes enabled us to infer the relative presence of each subunit in the global cell population of both PPN and LDT. Overall, GABAAR γ2 subunit (Fig. 6, gray middle circular crown) is present in 87% of PPN cells but only in 65% of LDT cells. This difference is due on one hand, to the fact that the percentages of both glutamatergic and GABAergic cells co-expressing the GABAAR γ2 subunit in the PPN were higher than those in the LDT (Table 5). In addition, the percentage of co-expression of the γ2 subunit in the glutamatergic cells was higher than in GABAergic cells in both nuclei (Table 5), and the former subpopulation is larger in the PPN than in the LDT, as previously reported (Luquin et al. 2018).

Fig. 6
figure 6

Estimated percentage of the GABAAR γ2 and GABAB R2 subunits in the total PPN and LDT, with respect to the relative percentages of the three cell phenotypes in each nucleus. The outermost circular crown represents the percentual estimations of glutamatergic (blue), cholinergic (red) and GABAergic (green) subpopulations in the PPN (a) and LDT (b), as previously estimated (Luquin et al. 2018); the middle one (solid gray) represents the estimated percentage of co-expression of GABAAR γ2 in the three subpopulations in the PPN (a) and LDT (b), whereas the innermost circular crown (gray-lined) represents the estimated percentage of co-expression of GABAB R2 in the same subpopulations of the two nuclei

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In contrast, the percentage of GABAB R2-expressing cells in the PPN and LDT (shaded innermost circular crown) was very similar (70 and 68%, respectively), as the percentages of co-expression of the R2 subunit in each phenotype were virtually the same in the two nuclei.

Discussion

Here we have provided the first anatomical evidence of the presence of GABAAR and GABAB receptors in the different cell phenotypes in the PPN and LDT. A detailed quantitative analysis of GABAAR γ2 and GABAB R2 subunits in the two nuclei was carried out, and the mean total number of cells expressing each subunit was estimated. Furthermore, we also quantified the number of cells expressing each receptor subunit within each neurochemical subpopulation. While all cholinergic cells in both nuclei expressed GABAAR γ2 and GABAB R2, only a certain proportion of the total glutamatergic and GABAergic cells expressed the γ2 subunit, suggesting that two functionally heterogeneous subsets of cells may coexist among cells of the same phenotype. The proportion of GABAAR γ2-expressing cells in the two non-cholinergic cell subpopulations differed between the PPN and LDT. This, together with a marked difference between the two nuclei regarding the presence of GABAAR α1 subunit in the cholinergic cells might contribute to functional differences between the PPN and LDT.

Methodological considerations

All antibodies used had been characterized previously (see “Experimental procedures”). Immunohistochemical detection is known to critically depend on epitope accessibility (Lorincz and Nusser 2008). In our colorimetric sections epitope accessibility was confirmed, as all α1-, γ2- and R2-immunolabelings were observed at all levels of the z-stacks, ensuring that the antibody had penetrated completely throughout the sections. However, for fluorescence immunolabeling of α1 and R2 subunits, a pretreatment of water-bath heating was carried out for antigen retrieval, to enhance the labeling (see “Experimental procedures”). Immunoreactivity against α1 was still light, but a low affinity of the α1 antibody was disregarded, as intense α1-ir was observed in the median raphe nucleus and ventral tegmental area in the same sections as previously reported (Fritschy and Möhler 1995; Pirker et al. 2000), confirming the specificity and sensitivity of the antibody used here. Such low immunoreactivity against α1 may reflect the low presence of this subunit in cell bodies of the PPN and LDT, as reported (Fritschy and Möhler 1995), and may well account for its scarce or null detection (Pirker et al. 2000; Rodríguez-Pallares et al. 2001).

GABAAR γ2 subunit in PPN and LDT

Whole-brain immunocytochemical studies reported the presence of immunoreactive cell bodies expressing diverse GABAAR subunits in the PPN and LDT (Fritschy and Möhler 1995; Pirker et al. 2000), while pharmacological and electrophysiological studies specifically showed the presence of functional postsynaptic GABAA receptors in the two nuclei (Kohlmeier and Kristiensen 2010; Ikeda et al. 2004; Saitoh et al. 2003). The present results confirm the findings of the electrophysiological studies regarding the presence of GABAA receptors in PPN and LDT (Kohlmeier and Kristiensen 2010; Ikeda et al. 2004; Saitoh et al. 2003); moreover, and consistent with Fritschy and Möhler (1995), these results confirm the presence of somatic GABAAR α1 and γ2 subunits in the PPN and LDT and extend the anatomical data estimating that 87% of PPN cells and 65% of LDT cells specifically express the γ2 subunit (Fig. 6).

Regarding the neurochemical phenotype of GABAAR γ2-expressing cells, we have shown that all mesopontine cholinergic cells express GABAAR γ2. The specific presence of functional GABAARs in mesopontine cholinergic cells was reported first in the PPN (Saitoh et al. 2003) and then in the LDT (Kohlmeier and Kristiensen 2010); more recently, indirect anatomical evidence was provided by the beautiful demonstration of GABAAR γ2-ir in cholinergic axon terminals in the pontis oralis nucleus (Liang and Marks 2014), most likely originating from cholinergic PPN and LDT neurons (Mitani et al. 1988; Semba et al. 1990; Shiromani et al. 1988). Consistent with this, we have confirmed the presence of GABAAR in the cholinergic subpopulation of the PPN and LDT, and specifically of the γ2 subunit (Liang and Marks 2014), and have extended these data to the 100% of cells.

GABAAR subtypes can be roughly subdivided into those preferentially located at synaptic sites versus those located extrasynaptically. The ones located synaptically are those containing mainly the α1, α2, and α3 subunits, together with β subunit variants and the γ2 subunit (Fritschy and Panzanelli 2014), being the γ2 subunit essential for synaptic localization and clustering of the receptor subtypes (Essrich et al. 1998). At the same time, synaptically located GABAAR subtypes undergo phasic activation and are responsible for fast inhibition. Our results indicate that all cholinergic neurons are endowed with GABAARs mediating fast inhibition. This does not preclude that additional GABAARs containing γ1, γ3 or δ subunits associated to subtypes located extrasynaptically, might also be present in this cell phenotype. Actually, the presence of the δ subunit in LDT cholinergic cells has been strongly suggested after electrophysiological studies (Kohlmeier and Kristiensen 2010).

Regarding non-cholinergic cells, 10% of glutamatergic neurons in the PPN and 35% in the LDT were γ2-negative, while 23% (PPN) and ~50% (LDT) of the GABAergic ones also lacked γ2. The γ2 subunit is associated with the vast majority of GABAAR subtypes (Fritschy and Möhler 1995) while receptors in which the γ2 is replaced by γ1, γ3 or δ are less abundant. However, single cell bodies expressing the γ3 subunit and moderate numbers expressing the δ subunit have been reported in the PPN (Pirker et al. 2000), suggesting that γ2-negative glutamatergic and GABAergic cell subsets might instead contain either γ3 or δ subunits. Thus far, GABAAR subtypes containing the δ subunit have been predominantly or exclusively found at extrasynaptic sites in cerebellar granule cells and dentate gyrus granule cells (Nusser et al. 1998; Wei et al. 2003). Altogether, the present findings suggest that while a substantial proportion of glutamatergic and GABAergic cells in the PPN and LDT express synaptically located GABAAR subtypes, a proportion ranging from 10 to 50% of the two cell phenotypes does not, likely expressing extrasynaptic GABAARs only. Two subsets of GABAergic cells would thus be present in PPN and LDT: a larger one with cells subject to fast inhibition, and a smaller one, subject to tonic inhibition-mediated control; likewise, a similar segregation would be present among glutamatergic cells as well.

GABAARγ1 subunit in PPN and LDT cholinergic subpopulations

In addition to the γ2 subunit, co-expression of the α1 subunit was also analyzed in the cholinergic subpopulation. Moderate or intense GABAAR α1-ir had been reported in cell bodies of the LDT or PPN, respectively, as well as moderate α2- (PPN) and α3-ir (Fritschy and Möhler 1995). Here, our immunofluorescence analysis revealed that 50% of PPN cholinergic cells and 11% of LDT ones co-expressed the α1 subunit. GABAARs contain two α subunits that can be homologous or heterologous. While the α1α1-containing receptor is by far the most abundant GABAAR subtype in brain (Benke et al. 2004), other combinations, like α1–α3, α1-α2, α3–α3, and α2–α2 coexist in native receptors (Araujo et al. 1996; Benke et al. 2004). Dual immunocytochemical studies have shown that ChAT-positive neurons in the PPN express the α3 subunit, while some scattered cholinergic cells also expressed α2 (Rodríguez-Pallares et al. 2001). Furthermore, cholinergic interneurons in the striatum, septum, diagonal band of Broca and PPN are all α3–positive (Gao et al. 1995; Rodríguez-Pallares et al. 2000, 2001). According to this, it seems plausible that cholinergic neurons express common GABAAR subtypes comprising specific subunits. A detailed study of the expression of GABAAR α1−α3 subunits in mouse cholinergic striatal interneurons has reported that the α3 represented 46% of the total a subunit content, α1, 37% and α2, 17% (Boccalaro et al. 2019). This distribution would be consistent with the reported predominance of α3 in GABAAR in rat cholinergic neurons (Gao et al. 1995; Rodríguez-Pallares et al. 2001), and compatible with a relevant proportion of α1 as well, as reported here, wherein 50% of PPN cholinergic neurons expressed the α1 subunit. Altogether, we may hypothesize that 50% of PPN cholinergic neurons likely contain GABAARs comprising either an α1–α1 combination (generally more frequent, according to Benke et al. 2004) or an α1−α3 one (more likely), whereas the remaining 50% would instead comprise either the α2−α3 combination, or the α3−α3, consistent with the reported data (Fritschy and Möhler 1995; Pirker et al. 2000; Rodríguez-Pallares et al. 2001).

Distinct subunit composition confers specific deactivation and desensitization properties that may profoundly affect synaptic decay kinetics and the capability to sustain high frequency synaptic inputs (Tia et al. 1996). Actually, the presence of α1 subunit confers a decay of only a few milliseconds to α1 subunit-containing GABAARs (Bartos et al. 2001), but of tens of milliseconds to α3 subunit-containing GABAARs (Eyre et al. 2012). Cells that express GABAARs subtypes containing heterologous α subunits—as seemingly do PPN and LDT cholinergic cells, show a variability in decay kinetics that parallels the α1/α3 ratio in a given cell that would lay somewhere between the extremes seen with α1-only- and α3-only-expressing cell types (Eyre et al. 2012). The fact that a variable proportion of PPN and LDT cholinergic neurons contain at least one α1 subunit while the remaining cells do not, suggests that at least two cholinergic subsets would be present in both nuclei, one endowed with a faster synaptic decay than the other. Differences in the electrophysiological properties of PPN/LDT cholinergic cells have been consistently reported (i.e., Boucetta et al. 2014; Mena-Segovia et al. 2008; Sakai 2012; Takakusaki et al. 1997). It is thus plausible to hypothesize that, differences in GABAAR receptor kinetics between the two subsets may somehow contribute to the overall electrophysiological differences found among these cholinergic cells: to what extent and how, remains to be investigated.

GABAB receptor in the PPN and LDT

Electron microscopic studies have shown that the two subunits of GABABRs are expressed both presynaptically and postsynaptically in most neurons of basal ganglia nuclei in rat and monkey (Boyes and Bolam 2007 and refs therein). Postsynaptically, both subunits were mainly located at extrasynaptic sites, although some of them were found at symmetrical synapses, likely co-localized with GABAARs (Boyes and Bolam 2007). Regarding the PPN and LDT, electrophysiological and pharmacological studies have evidenced the presence of postsynaptic GABABRs in the two nuclei (Heinmiller et al. 2009; Kohlmeier and Kristensen 2010; Kohlmeier et al. 2013; Ulloor et al. 2004), although to our knowledge, protein expression in the LDT has only been reported in a whole-brain analysis of GABABR-immunoreactivity using the R1 subunit (Margeta-Mitrovic et al. 1999). Here, in line with that study, we have confirmed the presence of postsynaptic GABABRs in the PPN and also in the LDT using the R2 subunit, further supporting that the two GABABR subunits are necessary for functional GABABRs (Benke et al. 1999; Bettler and Tiao 2006; Charles et al. 2001). In addition, we have extended those results determining that virtually all cholinergic cells, as well as two-thirds of the glutamatergic and GABAergic cells in both nuclei, express the R2 subunit.

GABAB receptors are negatively coupled to cyclase adenylate via Gi proteins. This accounts for the delay in the onset of the hyperpolarization mediated by these receptors as well as for their slow time course. As reported here, nearly all cholinergic cells express GABABRs receptors, in addition to the expression of synaptic GABAARs in 100% of them; these findings support that virtually all mesopontine cholinergic cells are not only under fast inhibitory control through GABAARs, but also under slow inhibitory control by GABABRs. Furthermore, the fact that virtually all mesopontine cholinergic neurons express both GABAARs and GABABRs, suggests that single cells contain the two receptor types. Interestingly, a recent study has shown a cross-talk between GABAARs and GABABRs in the mammalian central nervous system in which the activation of GABABR suppressed GABAAR responses, and this disinhibition occurred when both receptors were present on the same cell (Shen et al. 2017). While the outcome of either suppressing or enhancing inhibition of GABAARs may depend on the subunit composition of the GABAARs (Shen et al. 2017 and refs. therein), the same-cell localization of GABAARs and GABABRs in cholinergic cells suggested here provides the anatomical substrate for a potential effect of one receptor type upon the other, in addition to the individual function of each receptor type alone.

Regarding the non-cholinergic cells, two subsets of glutamatergic and GABAergic cells seem to also be present in PPN and LDT in relation to GABABRs, a larger one expressing the receptor, and a smaller one lacking the R2 subunit, and thus most likely lacking functional GABABRs. If we combine the expression of GABAARs and GABABRs (Fig. 6), glutamatergic cells in either nucleus might potentially be divided into up to four subsets according to their particular GABA receptor profile: GABAA + /GABAB + , GABAA + /GABAB−, GABAA−/GABAB + or GABAA−/GABAB−. Similarly, up to four subsets of GABAergic cells may potentially exist. The differential GABA receptor profile of each subset most likely confers particular electrophysiological properties to the subset within each cell phenotype. Electrophysiological studies in vivo in the PPN have reported a variety of firing patterns in non-cholinergic cells, corresponding to both presumed excitatory and inhibitory cells (Mena-Segovia et al. 2008; Ros et al. 2010; Sakai 2012); in one of those studies, at least three different functional subsets were identified: tonically firing, irregular, and quiescent neurons which fired in different phases of cortical activity (Ros et al. 2010). In addition, heterogeneous responses were also observed when recording from GABAergic neurons in the same region during spontaneous locomotion, “indicating additional complexity in the composition and function of this subpopulation” (Roseberry et al. 2016). How the diversity in GABA receptor profiles among glutamatergic and GABAergic cells and their particular dynamics might contribute to the electrophysiological differences observed among those cell subpopulations deserves further research.

Differential expression of GABA receptors in the PPN and LDT

Some of the data reported here revealed relevant differences between the PPN and LDT. One is the substantially lower proportion of α1 subunit in LDT cholinergic cells (11%) compared to PPN (50%), which would imply that nearly 90% of LDT cholinergic cells contain GABAARs comprising either α2–α3 or α3–α3 combinations. The deactivation kinetics of α1β2γ2 recombinant receptors are several fold faster than those of α1β2γ2 ones (Barberis et al. 2007), differences that were replicated in thalamic neurons expressing predominantly the α1 subtype (ventrobasal complex, VB) or the α3 (reticular nucleus, RT) (Mozrzymas et al. 2007 and refs. therein). Furthermore, these differences accounted for the rapid versus slow synaptic currents elicited in the VB and RT cells, respectively; however, the physiological role of these differences between inhibitory postsynaptic currents is not clear (Mozrzymas et al. 2007). These authors suggest that faster inhibitory postsynaptic currents elicited in α1-containing cells make them more appropriate for tasks requiring a high temporal resolution, whereas extremely long lasting inhibitory currents such as those elicited in α3-containing cells, provide a particularly potent mechanism for inhibitory drive (Mozrzymas et al. 2007). How rapid versus slow inhibitory postsynaptic currents elicited in PPN and LDT cholinergic cells, respectively, might affect their overall activity and on a much larger scale, might contribute to their segregated functions (** analysis. Front Neuroanat 10:1" href="/article/10.1007/s00429-021-02375-9#ref-CR20" id="ref-link-section-d9800391e3471">2016a,b). However, the neural circuits within the PPN and LDT remain largely unknown. While the cholinergic and glutamatergic subpopulations seem to have a larger amount of cells directly targeting them from the diverse basal ganglia components (Roseberry et al. 2016; Caggiano et al. 2018; Henrich et al. 2020), the cell-specific targets of nigrotegmental and pallidotegmental projections and their relative distribution among the three cell phenotypes are only partially known. Electrophysiological and pharmacological studies showed that 30% of PPN neurons contacted by nigral fibers were cholinergic, and that all inhibitory postsynaptic potentials evoked by nigrotegmental projections were controlled via GABAARs (Saitoh et al. 2003; Ikeda et al. 2004). In addition, PPN non-cholinergic cells targeted by nigrotegmental projections have been identified as glutamatergic cells (Grofova and Zhou 1998). The data reported here that GABAARs are present in the three cell phenotypes confirms that an inhibitory control may be exerted by nigrotegmental projections on both cholinergic and glutamatergic cells, and to a minor extent, on GABAergic cells (Roseberry et al. 2016). In addition, work from our laboratory has revealed potential synaptic contacts of pallidotegmental fibers arising from both the entopeduncular nucleus and the ventral pallidum onto cholinergic, GABAergic, and glutamatergic cells in the PPN and LDT (Mongia et al. 2015; Mongia 2016); interestingly, the relative proportion of contacts on cholinergic cells was also one-third of them, similarly to the reported electrophysiological data on nigrotegmental projections (Saitoh et al. 2003). Although to our knowledge the receptors implicated in these projections have not been established, we may hypothesize that they will also act via GABAARs receptors.

As far as we know, there is no physiological evidence of what afferent projections may be eliciting GABAB-mediated responses in PPN or LDT in relation to basal ganglia downstream projections. However, there are other GABAergic inputs to the PPN and LDT that could be acting on these receptors, arising from structures that participate in different functional circuits (Roseberry et al. 2016; Caggiano et al. 2018; Henrich et al. 2020). For example, a large number of neurons from the central amygdala and the bed nucleus of the stria terminalis, structures involved in fear-related behavior, heavily innervate both nuclei. Actually a number of excitatory, putative glutamatergic cells in LDT are inhibited via GABABR stimulation originating in local GABAergic interneurons, leading to freezing behavior (Yang et al. 2016). Also, GABAB receptors in the PPN mediate suppression of REM sleep and suppress unitary activity of cholinergic cells which are REM-active in the freely moving rat model (Ulloor et al. 2004). Specifically, GABAergic inputs from the ventrolateral periaqueductal grey controlling PS are projecting to cholinergic and glutamatergic neurons in the ventral LDT (Sapin et al. 2009; Weber et al. 2018). Whether these projections and/or additional local GABAergic neurons (Kroeger et al. 2017) act via GABAB receptors, is something that remains to be determined.

Conclusions

Downstream projections from the basal ganglia output nuclei directly target the three cell phenotypes in the PPN and LDT, and as we show here, they are all endowed with GABA receptors enabling GABAergic transmission. What specific cell subsets of cholinergic and non-cholinergic cells are actually controlled by these projections, and what local and/or reciprocal connections to basal ganglia components do they establish, is a matter of future research that will contribute to understand the role of PPN and LDT within basal ganglia circuitry.

Marked differences were found between the PPN and LDT in relation to GABAAR subunit composition in the cholinergic subpopulation, as well as to the synaptic versus extrasynaptic GABAARs in their non-cholinergic subpopulations. The significance of GABAAR heterogeneity for brain function is not yet understood. What we know is that the diverse dynamic characteristics of different GABAAR subtypes give rise to distinct types of inhibitory control, some of which may coexist in a given nucleus (Ye et al. 2017), while the presence of GABABRs likely provides additional types of inhibitory control. Additional work on GABAAR subunit combinations and their particular kinetics, as well as on their potential interactions with GABABRs will help understand inhibitory neurotransmission in these nuclei and may lead to a better understanding of functional differences between the PPN and LDT.